By using cells overexpressing diphtheria toxin (DT) receptor and a novel method of permeabilizing the plasma membrane with a bacterial pore-forming toxin, specific translocation of fragment A to the cytosol was observed, whereas full-size DT and other minor species of DT-derived fragments were left in intracellular vesicles. The translocation competence of DT proteins with mutations in the transmembrane domain is consistent with their cytotoxicities. The charge-reversal mutants E349K and D352K do not translocate their fragment A to the cytosol, whereas D352N is partially competent. ADP-ribosyltransferase activity of fragment A is not required for translocation. Novel fragments of DT with apparent molecular masses of 28 and 35 kDa were detected in endocytic vesicles. The 28-kDa fragment consists of fragment A and an N-terminal piece of fragment B, whereas the 35-kDa fragment contains part of fragment B and may be complementary to the 28-kDa fragment. Time course studies show that the 28-kDa fragment appears in endocytic vesicles prior to translocation of fragment A to the cytosol, raising the possibility that the 28-kDa fragment is an intermediate in translocation. We present a model for translocation of fragment A that incorporates the observations made using the novel permeabilization method.